Thiamin content of three sources of corn and arepas as determined chemically and microbiologically

THIAMIN CONTENT OF THREE SOURCES OF CORN AND AREPAS
AS DETERMINED CHEMICALLY AND MICROBIOLOGICALLY

by

LAURA LEE KELLER

B.S., Kansas State University, 1979

A MASTER'S THESIS

submitted in partial fulfillment of the

requirements for the degree

MASTER OF SCIENCE

Department of Foods and Nutrition

KANSAS STATE UNIVERSITY
Manhattan, Kansas

1985

Approved by:

LtXsvd:i- U. ■?. /Gb-cJ?*

'y-tsLd^

ins

TABLE OF CONTENTS

A1150B miMDS

PAGE
i v

LIST OF TABLES

LIST OF FIGURES v

INTRODUCTION 1

REVIEW OF LITERATURE 4

Corn: history, use, and types 4

Origin and use of the arepa 8

Types and preparation of arepas 9

Nutritional studies on arepas and tortillas. .... 10

Thiamin 13

Thiamin assays 15

Microbiological vitamin assay studies. . . 19

MATERIALS AND METHODS 21

Source of materials. 21

Preparation of arepas 22

Flour particle size, pH, moisture 23

Chemical analysis. ...... 24

Microbiological analysis . . . . . . . 24

Statistical analyses . . 24

RESULTS AND DISCUSSION 25

Percentage moisture 25

pH 26

Thiamin in corn . . . . 29

Thiamin in arepas - chemical measurement 30

Percentage thiamin lost in arepa preparation 30

i i

Thiamin in arepas - microbiological measurement. . . 33

Chemical and microbiological method differences. . . 33

Personal observations 35

Recommendations 35

SUMMARY 36

CONCLUSIONS 37

REFERENCES 38

ACKNOWLEDGEMENTS 41

APPENDIX 42

i i i

LIST OF TABLES

TABLE PAGE

Table 1. Maize diets in selected countries, g/caput/day. 5

Table 2. Calorie and nutrient intake levels of maize

diets in selected countries, intake/nutrition
unit/day 7

Table 3. F-values for variables among sources. ..... 25

Table 4. Mean percentage moisture, thiamin loss, and pH

change values among sources ... 26

Table 5. Analysis of variance for differences:

A. of thiamin amounts among corns

B. between two methods among sources. ..... 29

Table 6. Raw data for corn: thiamin and pH measurements. 43

Table 7. Raw data for arepas: chemical, microbiological,

pH, and moisture measurements 44

i v

LIST OF FIGURES

FIGURE

PAGE

Figure 1 .
Figure 2 .

Mean pH values of corns and arepas

Mean thiamin amounts of corns and arepas as
measured chemically and mi crobiol ogi cal ly.

28

32

1

INTRODUCTION

An arepa is an unleavened round flat bread made from
corn flour, water, and salt. Arepas are known as the
national bread of Venezuela, being consumed by 90% of the
population; however, arepas also are eaten in other Latin
American countries [1], Arepas are nutritionally deficient,
especially in lysine, thiamin, vitamin C, and iron [2].
Nunez and Maga [2] cited a pamphlet published by Consejo
Nacional de Investigaciones Cientificas y Technologicos de
Venezuela (CONICIT) in 1976 stating the nutritional and food
technological priorities for Venezuela. Included in those
was the development and supplementation of cereal products
and the development of new products to meet the Venezuelan
nutritional needs. As a primary cereal dish, arepas are the
main source of thiamin for the Venezuelan population. There-
fore, improvement of the thiamin content of arepas would
fulfill a priority as stated by CONICIT.

White dent is the most common type of corn used in
making corn flour for arepa preparation [1]. The primary
reason for arepas' thiamin deficiency is the method of pre-
paring the corn flour. In various methods of arepa produc-
tion the pericarp, and often the germ, are lost; and since
these are the principal thiamin sources of the corn kernel,
the thiamin content is reduced. Sometimes the corn is soaked
in an alkaline solution, thus the thiamin content is reduced
in these arepas since thiamin is destroyed by alkali. Altei —

ing the method of preparation could decrease thiamin loss,
but the resulting arepa likely would not be as acceptable to
people accustomed to arepas prepared in traditional ways.
Comparison of research on arepas shows that thiamin content
of corns vary somewhat depending on the source, but a com-
parison of thiamin content of various corns could not be
found in research literature.

Most thiamin determination relating to corn products is
done by chemical assay. In their thiamin determination of
arepas, Suarez [1] and Jaffe [3] used chemical assays; and
Bressani et al. [4] chemically determined the amount of thi-
amin lost from corn to tortilla. None of these researchers
determined the amount of thiamin lost from corn to arepas
made from homemade masa. Gregory and Kirk [5] stress that
accurate chemical determination of the vitamin content of
foods is not very significant if the correlation between the
chemical value and biologically available amount of the vit-
amin in the food is not known. Determining the bioavailable
amount of vitamins in food is important in determining the
adequacy of dietary intakes. Gregory and Kirk [5] used rat
bioassay to determine bioavailability of thiamin in foods.
Bioavailability of vitamins usually is determined micro-
biological 1 y. No research on mi crobi ologi cal ly measuring
the thiamin content of corn or arepas could be found.

The purpose of this research was to prepare arepas by a
traditional method using white dent corn from three different

geographical regions: Kansas, Mexico, and Venezuela. The
amount of thiamin in the whole corn was determined chemi-
cally, and the amount of thiamin in the arepa was determined
by chemical and microbiological assays.

The data obtained were analyzed to determine

1) if there was a significant difference in thiamin
content of the different corns used,

2) how much thiamin was lost during arepa prepara-
tion as measured chemically,

3) how much thiamin was bioavailable in the arepa
as measured by microbiological assay, and

4) if there was a significant difference in the
thiamin levels measured by the two different
methods of analysis.

REVIEW OF LITERATURE
Corn: History, Use, and Types

Corn is a plant with several common names. Maize is
the most widely used and international term; but in the U.S.,
maize's equivalent term is corn, thus that is the the term
used with this research project. Zea mays L. was given to
corn in the 18th century as it's botanical name. Zea is
Greek for cereal, being derived from a verb meaning "to
live", and mays is of Indian origin. The importance of corn
in many populations' diets is shown by the fact that many
Indian forms of the word corn meant "that which sustains us"
[6]. Corn's probable origin is the Americas, the central
and/or southern regions. The earliest ears of corn found
were discovered in Mexico. They were small ears dating back
to 2000 B.C. and were from a cultivated plant [7].

Although corn production has spread worldwide, countries
differ in how much corn and corn products they consume.
"Maize eating" populations are countries for whom maize is
the staple food, and are usually in regions which are poor
and less developed agriculturally. There are a number of
these countries, but Tables 1 and 2 refer to four of the main
ones; Romania, Mexico, Venezuela, and Guatemala [8]. Al-
though this information is over 30 years old, it is useful
in presenting a general idea of the importance of corn
products in Venezuela and the other countries. Table 1

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describes the diets of the various groups in the countries.
Table 2 shows the calorie and nutrient intakes per nutrition
unit per day of the countries compared to the RDA for an
adult man. These figures reveal that the diets and nutrient
intakes vary with the country and with the groups of people
within the country. The poorer the people, the less nutri-
tionally adequate their diet. In 1950, Venezuela was the
only country whose daily nutrition unit intake for thiamin
was lower than the RDA. The World Health Organization's 1967
nutrition intake statistics revealed that the total thiamin
intake in Venezuela that year was 0.69 to 0.80 mg/person/day
[9], The Recommended Daily Allowance for an adult male is
0.5 mg/1000 kcal [10]. More recent information could not be
found, but this information illustrates the need for increas-
ing thiamin intakes for Venezuelans.

The main types of commercial corn are: dent, flint,
floury, sweet, and popcorn. Dent is used in the preparation
of arepas, and it can be white or yellow. White corn is
almost always used for making arepas. Yellow corn is not as
abundant as white in Venezuela and is more expensive; but
yellow corn occasionally is used to make an arepa which is
different in flavor and appearance, higher in niacin and
carotene, and yet acceptable [1].

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8

Origin and Use of the Arepa

Suarez [1] and Cuevas et al. [11] discussed the fol-
lowing history and use of arepas. The arepa probably ori-
ginated with the first known Indians in Venezuela, and other
corn dishes followed. The original name "EREPA" was the
generic name of ripe corn in the Indian language Cumana-
goto. In other countries the term arepa is used for a corn
cake slightly different from the type made in Venezuela. The
Indians shaped the arepa round to resemble the sun. The
arepa always has been the primary bread of Venezuela, and no
promotion to eat arepas has been needed; it is popular on its
own. Arepas often become popular with foreigners who are
visiting or staying in Venezuela.

The arepa is eaten by poor and wealthy alike. Once
arepas only were eaten at home; but now arepas are sold at
restaurants (areperias), schools, and other public places.
Arepas usually are consumed for breakfast, lunch, or snack,
but can also be eaten for dinner. They are eaten plain or in
combination with cheese, butter, eggs, red meats, poultry,
beans, jams, or other ingredients. Venezuelans eat an aver-
age of 3 to 4 arepas/day, or 155 g/day [1,11].

Arepas vary in size, shape, and thickness. Arepas can
weigh from 15 g to 150 g or larger, be flat or slightly
spherical, and range from very thin to thick. They can be
boiled, baked, fried, or a combination of these, or can be

toasted. Food may be eaten on top of them, or the arepa can
be spl it and f i 1 led [ 1 ] .

Types and Preparation of Arepas

When reading research articles concerning arepas, one
should realize that there are different types of arepas based
on corn flour preparation. The corn may be totally degermed,
partially degermed, or the germ left intact, and may be
soaked in water, calcium, ash, or other alkaline substances
[1]. The method of preparation will affect the appearance,
consistency, and nutritional value of the arepa.

Suarez [1] and Cuevas et al. [11] described the pre-
paration steps of a common traditional type of arepa in
Venezuela. The corn is degermed mechanically and soaked in
water. Following are the steps:

1) mechanical phase — the pericarp and germ are sepa-
rated and discarded by pounding the corn with a
mortar in a wooden bowl (pilon), or an electric
machine. The resulting pieces of corn look pol-
ished and are called pilado. Though the pilado
is supposedly degermed, it can retain up to 40%
of the germ [12].

2) wash.

3) cook — pilado is boiled in water to soften the
grain.

4) mill — the soaked pilado is ground with a metal or
stone mill. As the pilado is ground, water is
added to make a masa (dough) which will stick to
the mill. The masa will be white in color and
semi hard.

5) knead — the masa is kneaded with water and salt to
the proper consistency. The person preparing the

10

masa pats it, and knows it is ready when it has
a characteristic sound or "ring".

6) shape — a portion of masa is shaped in the palm of
the hand by patting a ball into a flat circle.

7) cook — the arepas are cooked on a hot plate or
grill called a budare. The budare is clay or
iron. The clay budare and wood oven give the
best flavor. The arepa should be golden brown
and the sides should be hard.

Arepas also can be prepared from non-degermed corn which

is cooked and steeped in an alkali solution to facilitate

removal of the pericarp. The alkaline soaked corn is called

nixtamal, and it is ground to form masa [1]. This masa can

be shaped as the masa previously described and cooked in the

manner desired.

Nutritional Studies on Arepas and Tortillas

Tortillas usually are prepared from nixtamal [3], thus,
nutritional research on these tortillas can be useful for
comparison with data on nixtamal-prepared arepa research.
These tortillas can differ from arepas in the type of corn
used and how they are cooked, so these differences, if pre-
sent, should be kept in mind when making nutritional compai —
i s o n s .

Early arepa research was conducted on homemade arepas.
Suarez [1] conducted nutritional studies on arepas made from
pilado, from nixtamal, and from yellow corn. Protein, fat,
carbohydrate, mineral, and vitamin content were determined.
Protein content was found to be low (3.95 to 5.50%); and the

11

B-vitamin, including niacin, amounts were low. Suarez sup-
plemented arepas with amino acids to improve protein effi-
ciency and prepared a supplemental mix of thiamin, ribo-
flavin, niacin, and iron to increase the arepa's nutritive
value. Jaffe [3] determined protein, fat, ash, fiber, cai —
bohydrate, mineral, and B-vitamin content of whole corn,
pilado, nixtamal, tortilla, and commercial arepa. Compared
to the nixtamal, the pilado had much more loss of fat, po-
tassium, thiamin, riboflavin, and niacin. The nixtamal was
considerably higher in calcium since it was soaked in a
cal ci urn sol uti on.

Chavez and Pellet [13] studied protein quality of 12
Latin American food mixtures using rat bioassay. They tested
five different arepa preparations including arepa with mai —
garine, arepa with white cheese, arepa with sardines, arepa
with black beans, and arepa with meat. The dry weight per-
cent protein of the arepas ranged from 8.2 to 22.0, and the
protein efficiency ratio (PER) ranged from 0.5 to 4.6.

Bressani et al. [4] studied chemical changes in white
and yellow corn during tortilla preparation. For the white
corn, the combined chemical and physical loss from corn to
masa averaged 60% thiamin, 52% riboflavin, and 32% niacin.

More recent research has been conducted on arepas made
from commercial corn flour. Nunez and Maga [2] compared
sensory properties of arepas made from packaged corn flour
and extruded corn flour. Seventeen panelists assessed the

12

arepas for acceptability, texture, and flavor; and signi-
ficant differences were revealed between packaged flour and
extruded flour. By comparing water absorption, water sol-
ubility, and consistency of a water suspension, the re-
searchers concluded that the extruded flour had better func-
tional characteristics than did the packaged flour.

Smith et al. [14] also produced corn flour using an
extrusion cooker, and compared arepas made from the extruded
flour to arepas made from flours from the conventional
column-roller process. Based on sensory and instrumental
tests they concluded that at appropriate extrusion condi-
tions, the extruded flour produced arepas equal to or better
than the conventional flour, thus extrusion could be a good
alternative to column-roller pre-cooking.

Alvarez [15] analyzed the proximate and mineral compo-
sition of baked and fried arepas from packaged corn flour.
The baked and fried arepas contained 54.81 and 26.82% mois-
ture, 0.74 and 13.26% fat, 4.26 and 6.28% protein, 1.09 and
2.03% ash, 0.83 and 0.85% fiber, 38.27 and 48.96% carbo-
hydrate, and 168.5 and 340.3 kcal/100 g, respectively. Zinc,
Mg, K, Cu, and Ca increased during processing, but no min-
erals were present in sufficient quantity to make the common
arepa a good source of any specific mineral. Water activity
and pH did not change significantly. In other research
Alvarez [16] studied the microbiological safety of baked and
fried arepas. One hundred and thirty-six bacterial and 65

13

fungal isolates were identified from the flour dough and
arepas; and although no pathogenic organisms were isolated in
the arepas, strict sanitary conditions were recommended to
achieve a safe product.

Harbers et al. [17] compared the quality of arepas made
from American midwestern grown corn to arepas made from
packaged corn flours. When compared to the doughs and arepas
prepared from commercial flours, the homemade masa was more
yellow; and arepas prepared from the homemade masa had a more
intense corn flavor and darker crust color, as determined by
a sensory panel and by instrument.

Cuevas et al. [11] reviewed traditional arepa prepai —
ation, development of precooked corn flour, and quality
assurance. Important factors in quality control included
hard endosperm corn, cooking and rolling conditions, and
particle size of flour. Optimum particle size for arepa
flour was listed as corresponding to screens between 35 and
48 mesh.

Thiamin

Chemical structure and properties. Thiamin is a water
soluble B-vitamin. Its structure is a simple compound com-
posed of a pyrimidine moiety and a thiazole moiety joined by
a methylene bridge [18].

14

H

HSC— C^JC— NHZ Hck-Ji— CHzCHzOH
H

m^c_chxOH Thiamin ff-lT^

HjC— C^i— NHZ Ht^l— CHiCH2OH

Pyrimidine Thiazole

Thiamin is found in natural material in the free form, as
mono-, di-f and triphosphoric esters, also as mono- and
disulfides. Free thiamin is the most abundant form found in
plant extracts. Below pH 5, thiamin is very stable. At pH
5 to 7, thiamin is destroyed by autoclaving, and above pH 7,
thiamin is destroyed by boiling or storage at room temper-
ature [19]. It is thought that the thiazole ring opens and
is oxidized, especially upon heating. In a highly alkaline
solution thiamine is oxidized by ferricyanide to thiochrome.

H Hi

H 3 °~^K^t^s^~C H* C H * ° H
Thiochrome

Food sources Good sources of thiamin include dried beans
and peas, nuts, whole grain cereals and breads, pork, and
organ meats. Dairy products, fruits and vegetables, and
other meats, poultry, and fish also contribute thiamin in
the diet [1], The recommended thiamin allowance is related
to energy intake; 0.5 mg/1000 kcal is the daily recommenda-
tion for adu Its [10].

15

Functions Several of the major functions of thiamin
include the body's use of thiamin to form the co-enzyme
thiamin pyrophosphate, which is important in energy meta-
bolism, and thiamin's action in nerve transmissions [20].

Deficiency symptoms When less than the minimum amount
of recommended thiamin is consumed over a period of time,
deficiency symptoms affecting the gastrointestinal, cat —
diovascular, and peripheral nervous systems appear [21].
Symptoms include loss of appetite, depression, and as defi-
ciency continues, constipation and neurological changes.
Beriberi is the severe form of thiamin deficiency, and chat —
acteristics include an enlarged heart, severe edema, and
muscle wasting [20,21].

Effects of cooking Thiamin loss from foods is caused by
cooking in water, heat, oxidation, and cooking in an alka-
line solution [20]. The time of heating, processing, and
storage also are important factors contributing to foods'
thiamin loss [22].

Thiamin assays

Thiamin in foods can be determined by the following
assays: animal, chemical, microbiological, high pressure
liquid chromotography, colorimetric measurement, and mea-
surement of absorption spectrum [23]. The most frequently
used assays are the first three.

16

Animal assays The first methods used to determine
thiamin in food were animal assays. The advantages of animal
assays are that they are specific, measure the physiologi-
cally available thiamin amount, and do not require extraction
procedures. They are not commonly used today, however,
because of cost, time required, and lack of precision [23].
Chemical assay The thiochrome technique is the assay
most often used for determining thiamin in natural materials.
Thiamin is extracted from the sample by dilute acid hydro-
lysis and enzymatic digestion. The pH of the acid extract
ensures that the thiamin is very stable, even when heated.
The enzyme solution used contains phosphatase which hydro-
lyzes any phosphate esters of thiamin present, converting
bound thiamin to its free form. The enzymes hydrolyze the
starches in plant samples, aiding extract filtration. The
thiamin present in the extract is oxidized to thiochrome by
alkaline ferricyanide, and extracted by isobutanal. Thio-
chrome has an intense blue fluorescence under ultraviolet
light, thus a photof 1 ourometer is used to determine amount
of f 1 ourescence; and thiamin amount is calculated. Several
disadvantages of the thiochrome method include its lack of
sensitivity and possible interference by other fluorescent
substances [19],

Microbiological assay Accurate chemical determination
of vitamin content of foods is of little value unless the
chemically assayed value can be correlated to the biologi-

17

cally available amount of vitamin in the food. Bioavail-
ability of vitamin data is important in evaluating the
adequacy of dietary intakes [5]. Compared with chemical
methods, microbiological methods for thiamin require less
equipment and material for assay, can be more sensitive and
specific, and many samples can be assayed inexpensively in a
short time. These methods are quite sensitive to thiamin
(0.001 mp»g to 1 jig) and generally are reproducible to better
than ±10%. However, microbiological methods can suffer from
poor reproducibility with slight variations in procedure or
if nonchemical ly defined media are used [18,23].

Microbiological methods for vitamin analysis are based
on the observation that certain microorganisms require spe-
cific vitamins for growth. When the samples containing the
vitamin are added to a nutrient medium and then inoculated
with the specific bacteria, growth over a specified incu-
bation time will be directly proportional to the amount of
vitamin present. Growth is measured photometrically, and the
sample solution and reference solution can be compared
accurately [24].

The following characteristics are required for a test
organism: require the vitamin, be genetically constant during
prolonged subculture, have a growth response that is easily
measured, have a rapid growth cycle, possess nutritional
requirements similar to man, and be non-pathogenic. Lacto-
bacilli are the microorganisms most often used. Yeasts,

18

molds, and protozoa are not used as frequently because their
growth characteristics usually are less suitable [19].

There are five types of thiamin requiring microorgan-
isms, differentiated by the type of thiamin they require.
They can require intact thiamin, the pyrimidine moiety, the
thiazole moiety, either the pyrimidine or thiazole moieties,
or both the pyrimidine and thiazole moieties. Mammals re-
quire intact thiamin, thus the microorganism used for thiamin
assay should also have this requirement [18].

Lactobacillus f ermentum is a bacterium which requires
intact thiamin for growth. This bacterium is rod shaped,
variable in size, non-motile, and heterof ermentati ve. There
is no growth at 15°C; optimum growth occurs at 41-42°C;
growth can occur at 45°C. Niacin also is required for growth,
but riboflavin and folic acid are not [25]. U_ fermentum can
be affected by pentoses, reducing agents, fructose, maltose,
calcium, and glucose heat degradation products. If the
incubation period is limited to 18 hrs, the pyrimidine and
thiazole moieties do not permit growth [23]. In 1944 Sarett
and Cheldelin introduced the use of L;_ fermentum for thiamin
determination. It is extremely sensitive for thiamin and is
not affected by thiamin moieties. It has been considered the
best bacteriological method for thiamin and has undergone
improvement over the years [23], Lactobaci 1 1 us viridescens
is another bacterium frequently used for thiamin assay. L.
viridescens is said to compare favorably with the thiochrome

19

lethod but is more specific and convenient [23].

Microbiological vitamin assay studies

Voight et al. [26] compared protozoan and conventional
methods of vitamin analysis. Thiamin content of tomato
juice, orange juice, blood, yogurt, round steak, and spinach
was determined with the bacteria Lactobaci 1 lus viridescens,
the protozoa Ochromonas dani ca, and the thiochrome method.
Except for tomato juice, all samples indicated higher
(P <0.05) amounts of thiamin as measured by U_ viridescens
than by thiochrome measurements. Generally, all three
methods indicated increased amounts of vitamins in samples
when the food extracts received acid hydrolysis and enzymatic
treatments.

Voight et al. [24] used L_^ viridescens and CL_ danica for
thiamin determination in another study. Standard vitamin
calibration curves were prepared to determine minimum and
maximum vitamin concentrations that could be determined by
microorganisms and protozoa, and to determine the incubation
times needed for the growth responses to stabilize. From 0.2
to 10 ng per ml, the two assay methods for thiamin were found
to be equally sensitive. The JU viridescens assay was
limited to 16 to 18 hrs since longer incubation time allows
enzymatic digestion of the bacterial cells, resulting in
decreased absorbance. Although (h_ danica was found to have a

20

stabilized growth response, it required four more days for
incubation than was needed for U_ viridescens.

To determine the thiamin content of triticale, wheat,
and rye, Michela and Lorenz [27] used l^ fermentum for
microbiological assay. The bacteria were received and re-
hydrated just before the grains were analyzed so that the
culture did not need to be maintained over a period of time.
The researchers did this because occasionally the bacteria
can develop the ability to synthesize thiamin when the re-
commended culture maintenance procedure is followed. The
whole-grain wheat thiamin values were higher than previous
thi ochrome-determi ned values reported in the literature.
Thiamin amount of triticale was equal to wheat, and rye's
thiamin amount was significantly lower than that of wheat or
triticale. The microbiol ogical ly-determi ned thiamin amounts
for the grain samples were believed to be accurate. There-
fore, those higher thiamin values were attributed to envi-
ronmental and agronomic conditions under which the grains
were grown, and not to method differences.

No studies determining the thiamin amount in corn or
arepas by microbiological assay could be found; however, Wang
and Fields [28] used Pediococcus cerevisiae for lysine assay,
and Lactobaci 1 1 us pi antarum for tryptophan assay in
home-prepared tortillas enriched with germinated corn.
Germination increases the lysine and tryptophan amounts in
corn, which is deficient in these two amino acids. As mea-

21

sured microbiological ly, the lysine and tryptophan values
increased from 23 mg/g N and 3 mg/g N for nongermi nated corn
to 68 mg/g N and 26 mg/g N after germination. However, taste
panels found the tortillas made from lime-treated corn to be
preferred over the germinated corn-enriched tortillas.

MATERIALS AND METHODS

Source of materials

Preliminary research Preliminary work included chem-
ical thiamin determination of six types of Kansas white
hybrid corns. Their thiamin values (;ig/g, dry weight) were:
4.34, 4.63, 5.03, 5.49, 5.74, and 6.05, showing differences
among varieties. Small hybrid sample amounts did not allow
using any of these corns for arepa production.

Selection of corn Three different corns were selected
to compare the amount of thiamin yielded in the arepas made
from each corn. A mixture of white hybrid dent seed har-
vested in Kansas (White Hybrid Performance 1984) was obtained
from the Kansas State University Department of Agronomy; a
white dent corn was obtained from The International Center
for Improvement of Maize and Wheat (CIMMIT), Mexico City,
Mexico; and a third white dent corn was obtained from Vene-
zuela through International Multifoods, Minneapolis, Min-
nesota. These three corns were stored at 0°C until prepara-

22

tion of the arepas. Cracked and broken kernels were sorted
out by hand and discarded.

Preparation of arepas

For making arepas, 100 g of each corn were rinsed in
distilled water. A modification of a procedure developed by
Hendershot [29] for corn tortillas was used to prepare home-
made masa. Two g of calcium hydroxide were mixed with 280 ml
of distilled water and placed in a two-quart stainless steel
pan. The corn was added, the pan covered, and the mixture
heated to 90°C on a Roper gas range and held at a temperature
of between 85°C and 90°C for 40 min. The pan was removed
from the heat, and the corn mixture was steeped for 11 hrs.

After steeping, the liquid was drained. The corn was
placed in a metal sieve and rinsed with running tap water for
5 min to remove pericarp, then frozen for 24 hrs at 0°C. The
frozen corn was freeze-dried for 24 hrs. The freeze-dried
corn was ground using a KitchenAid mixer (Hobart Corp, Model
K5-A) with a grain mill attachment (Model GM-A) to produce
approximately 90 g of corn flour. Sixty g of the Venezuelan
flour was combined with approximately 70 ml of distilled
water, and 60 g of each of the Mexican and Kansan flours were
combined with approximately 65 ml of distilled water. Each
flour was mixed in a stainless steel bowl to produce approx-
imately 125-130 g of cohesive dough.

23

Each arepa was made by pressing 40 g of dough into a
6.3 cm (2 1/2 ") x 8.2 cm (3 1/4") round mold sprayed, with
vegetable spray. The arepas were cooked 10 min on each side
on an iron griddle which had been preheated for 10 min to
204. 4°C (400°F) on a Roper gas range.

Flour particle size

To separate and discard flour particles larger than
0.425 mm, each corn flour was sieved with a 42-mesh screen
using a Ro-Tap Testing Sieve Shaker (Model B) for 15 min
[11].

£H

pH was measured on duplicate samples of corn and arepas
following AOAC procedure [10]. Ten g of each sample were
mixed with 100 ml distilled water and allowed to set for 30
min. The supernatant was poured off, and after 10 minutes,
the pH of the supernatants was taken.

Moi sture

Percentages of total moisture of the raw corn, freeze-
dried corn, and the baked arepa were determined in a C.W.
Brabender Semi-Automati c Rapid Moisture Tester (Model SAS
577). Duplicate 10 g samples were held at 120°C for 60 min
before percentages were determined [17].

24

Chemical analysis

Amounts of thiamin in the raw corn and cooked arepas
were determined using the thiochrome method [31]. The raw
corn was ground with grain mill attachment, and 3.5 g samples
were analyzed. The arepas were blended in a Waring blender
(Model 8), and 5 g samples were used for analysis. Preli-
minary work indicated that the Decalso purification step was
not needed. Flourescence was determined using an electric
photoflourometer (PH Coleman, Model 12-C). Thiamin content
was determined on a dry weight basis from four replications
of the thiochrome procedure.

Microbiolgical analysis

Amounts of thiamin in arepas were determined using the
procedure for thiamin assay described in the Difco Manual of
Dehydrated Culture Media and Reagents for Microbiological and
Clinical Laboratory Procedures [32]. The filtrate obtained
during chemical analysis of cooked arepas was inoculated with
Lactobacil lus fermentum. incubated at 37°C for 17-18 hrs, and
turbidity was determined using a spectrophotometer (Bausch
and Lomb Spectronic 20) at a wavelength of 540 nu. Four
replications of the procedure were made for each type of
arepa.

Statistical Analyses

Data for thiamin amounts of corns and arepas (both
methods), pH values of corns and arepas, % moisture of are-

25

pas, thiamin loss during arepa preparation, pH changes, and
microbiological and chemical values comparisons were analyzed
by analysis of variance (ANOVA) for a split-plot design.
When the ANOVA procedure indicated differences in means,
Duncan's Multiple-Range Test was used to determine signifi-
cant differences [33].

RESULTS AND DISCUSSION

Percentage moisture

Moisture content was not significantly different among
arepas (Table 3). The actual moisture content of arepas is
presented in Table 4. These % moistures are within the range
of 44.48% to 60.50% which Suarez [1] listed for 16 types of
arepas.

Table 3. F-values for variables among
sources.

Variable F-val ues

Corn pH 1.08

Arepa pH 134.94***

Thiamin content

Corn 134.5***
Arepa

chemical 1227.56***

microbiological 764.91***

Percentage moisture 0.56
*** P<0.001

26

Table 4. Mean* percentage moisture, thiamin loss,
and pH change values among sources.

Percentage moisture

Percentage thiamin loss
from corn to arepa

Change in pH
(corn to arepa)

Corn Source
MEX KAN VEN

45.01 44.68 45.89

24.1 30.4 47.5

+1.00 +1.43 +2.12

MEX = Mexico KAN = Kansas VEN = Venezuela
*Mean of four replications

£H

The F-values for differences in pH among corns and among
arepas revealed no significant differences for corns, but did
show differences (P<0.001) among arepas (Table 3). The
Venezuelan arepa had the highest pH, while the Mexican arepa
had the lowest pH (Figure 1). Harbers et al. [17] found mean
pH of arepas from homemade masa to be 8.16, which is in the
range of pH values found in this study. For each corn, there
was an increase in pH from corn to arepa (P<0.05) (Figure 1),
and these increases differed among sources (P<0.001). As
illustrated in Table 4, the Mexican corn had the smallest pH
increase, while the Venezuelan corn was affected most by the
alkaline steeping.

Figure 1. Mean* pH values of corns and arepas.

*Mean of four replications

MEX = Mexico KAN = Kansas VEN = Venezuela

ABC-values with different letters among sources differ
significantly (P<0.05) as determined by Duncan's
Multiple-Range Test.

28

MEX

KAN

YEN

CORN SOURCES

29

Thiamin in corn

Table 5 shows that when data for thiamin content in corn
were analyzed, the only variable to show significant dif-
ference was source. Values among replications and between
samples did not vary. Thiamin content of raw corns differed
(P<0.001) (Table 3). The Mexican corn had the most thiamin,
and the corn from Venezuela had the least (Figure 2). With
the exception of Venezuelan corn, which contained much less
thiamin than the other two sources, these amounts are similar
to the findings of Bressani et al. [4] and Jaffe [3], who
reported 4.57 and 5.0 jig/g thiamin, respectively.

No information could be found on factors influencing the
thiamin content of corn. However, for several other grains,
researchers do not agree on whether thiamin is influenced
genetically or by environment [33]. Both factors likely are
important, and further research is needed.

Table 5. Analysis of variance for differences:

A. of thiamin amounts among corns

B. between two methods among sources

source of variation

replication

source

rep x source

sampl e

source x sample

mean square/significance
df A B

3
2
6
1
2

0.00

0.16

2.38***

26.56***

0.01

0.09

0.00

0.00

0.00

0.01

*** P<0.001

30

Thiamin in arepas - chemical measurement

The chemically assayed thiamin contents of the arepas
were different (P<0.001) (Table 3). As in the raw corns,
Mexican arepas showed the highest thiamin content, Venezuelan
arepas showed the lowest, and Venezuelan arepas differed the
most in thiamin content (Figure 2). Suarez [1] determined
the amount of thiamin in 16 types of arepas; the values
ranged from 0.27 jig/g to 2.79 jofl/g. Jaffe [3] reported 0.34
jjjg/g thiamin for commercial arepas. Thus, the Mexican and
Kansan arepa values were higher than previously reported
val ues.

Percentage thiamin lost in arepa preparation

For each source, thiamin amounts decreased from corn to
arepa (P<0.05) (Figure 2), and these decreases differed among
sources (P<0.001). Table 4 shows that the Mexican corn had
the smallest, and the Venezuelan corn had the largest amount
of thiamin lost during arepa preparation. An increase in %
thiamin loss during arepa preparation corresponds to an
increase in pH of the arepa. This may be attributed to
thiamin's decrease in stability as pH increases.

Using similar preparation techniques as the arepa,
Bressani et al. [4] found a 60% loss of thiamin from corn to
masa (tortilla dough), and Jaffe [3] showed a 34% loss of
thiamin from corn to tortilla. Variations in reported %

Figure 2. Mean* thiamin amounts of corns and arepas as
measured chemically and mi crobiol ogi cal ly.

*Mean of four replications

Corn & Arepa-chem = thiochrome assay
Arepa-micro = L^ f ermentum assay

MEX = Mexico KAN = Kansas VEN = Venezuela

ABC-values with different letters/numbers among sources
abc differ significantly (P<0.05) as determined by
123 Duncan's Multiple-Range Test.

32

Com-chem

6 ■

w

Arepa-chem
Arepa-micro

0)

V
0)

\

0)

3 •

2
<

I

»-2-

1 •

10

o

N

MEX

KAN

VEN

CORN SOURCES

33

thiamin loss during tortilla preparation could be due to
different types of corn, and/or different methods of pre-
paration which remove varying amounts of germ and bran, the
thiamin rich parts of the corn kernel.

Thiamin in arepas - microbiological measurement

Arepa thiamin contents as determined mi crobiol ogi cal ly
differed ( P<0. 001 ) (Table 3). The Mexican arepa had the
highest value, and the Venezuelan arepa had a much lower
thiamin value when compared to the other arepas (Figure 2).
The three types of arepas had differing amounts of thiamin
(Table 5). These results may reflect accurately the amount
of thiamin that is bioavailable in the arepas; but due to
variability of results possible with microbiological methods,
further research would be needed to determine conclusively
the amount of thiamin bioavailable in the arepas.

Microbiological determination of corn was not conducted
because the bioavailability of thiamin in raw corn was not
regarded as necessary information. However, with the high
arepa values from the microbiological method, having those
measurements would have been beneficial in interpreting the
microbiological values.

Chemical and microbiological method differences

Within sources, the arepa's thiamin content differed
(P<0.05) as measured by the two methods (Figure 2). When
chemical and microbiological assays were compared, the Mex-

34

ican and Kansan arepas showed an increase in thiamin amount
(63.3% and 42.4 %), while the Venezuelan arepa showed a
decrease in thiamin amount (-51.5%). Among the corns, those
differences between methods differed (P<0. 001 ) (Table 5).

The higher microbiological values agree with Michela and
Lorenz [27], who reported higher thiamin values for wheat,
triticale, and rye using L_;_ f ermentum than for thiochrome-
determined values. Voight et al. [26] claimed that when
compared to thiochrome values, L^ viridescens indicated
higher thiamin values for all samples except one, which
showed a lower value.

There are several possible reasons for the much lower
microbiological value of the Venezuelan arepa. As other
studies have shown, the microbiological method can give vai —
ied results since it is a sensitive assay, thus the lower
value could be attributed to variability within the micro-
biological method. The Venezuelan arepa may have contained
an unknown factor which inhibited growth of l^ fermentum.
The treatment of corn to prevent germination can inhibit
bacterial growth, but the Venezuelan corn was untreated. A
third possible reason for the lower thiamin value would be an
actual reflection of increased loss of thiamin in the Vene-
zuelan arepa. The Venezuelan corn showed the largest in-
crease in pH and the greatest % loss of thiamin in arepa
preparation (Table 4). Because the microbiological method
can be more sensitive than the thiochrome method, the L.

35

f ermentum assay may be showing a more sensitive measurement
of the amount of thiamin lost in the Venezuelan arepa. Addi-
tional research would be needed to draw further conclusions.

Personal Observations

During arepa preparation, the author noticed subjective
differences among the corns and arepas. When thiamin is
dissolved in a strong alkaline solution, it turns yellow and
then fades [34]. All three corns became more yellow when
soaked in the alkaline solution, but the Venezuelan corn's
color became a deeper yellow, which resulted in the Vene-
zuelan arepa being noticeably more yellow than the other two
arepas.

In addition to color, the Venezuelan flour had other
differing characteristics when compared to the Mexican and
Kansan corn flours. The Venezuelan flour required less water
to form a dough; the dough formed was less sticky and more
closely resembled commercial arepa dough. The Mexican and
Kansan corns produced whiter, more sticky doughs which were
harder to shape than were the Venezuelan arepas. All three
types of arepas required the same cooking time.

Recommendations

Little is known about how thiamin is determined in corn,
and few studies have determined corn's thiamin content.
Corns analyzed in preliminary research had varying thiamin
amounts. The three sources of corn from this study had dif-

36

ferent thiamin contents and had different changes in alka-
linity, resulting in varying thiamin loss during arepa pre-
paration. Therefore, research seeking to understand factors
affecting corn's thiamin content, and work on developing
corns with higher thiamin amounts that lose less thiamin
during alkaline soaking could help improve arepas' thaimin
val ues.

SUMMARY

White dent corns from Kansas, Mexico, and Venezuela were
used for arepa preparation from homemade masa. For each
source of corn, thiamin content was determined by the thio-
chrome method. Thiamin content of each type of arepa was
determined chemically and mi crobiologi cal ly. pH change and
thiamin loss from corn to arepa were calculated.

Data were analyzed by ANOVA using a split plot design.
When differences in means were indicated, Duncan's Multiple-
Range Test was used to determine significant differences.

Thiamin content varied among sources of corn. Measured
chemically, thiamin loss occurred from corn to arepa. When
compared to chemically determined amounts, microbiological
thiamin determinations resulted in higher values for Mexican
and Kansan arepas, and a lower value for the Venezuelan
arepas. The two methods gave differing amounts of thiamin
for each type of arepa.

37

The pH values increased from corn to arepa due to alka-
line steeping. Among sources during arepa preparation, an
increase in % thiamin loss corresponded to a higher increase
in pH value. Percentage moisture of arepas did not differ.

CONCLUSIONS
Under the conditions of this study,

1. Thiamin content among the three sources of corn
varied significantly; with Mexican corn having the
greatest amount, and Venezuelan corn having the least.

2. Thiamin loss during arepa preparation increased
proportionally to increased alkalinity of the arepa.

3. For arepas, microbiological assays gave
significantly different thiamin values from those of
chemical assays.

4. Among sources, those having higher thiamin content
for the corn resulted in higher arepa thiamin values.

38.

REFERENCES

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4. Bressani R, Paz Y Paz R, Scrimshaw NS (1958) Chemical
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5. Gregory JF, Kirk JR (1978) Comparison of chemical and
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27. Michela PP, Lorenz K (1976) The vitamins of triticale,
wheat, and rye. Cereal Chem 53:853-861

28. Wang Y, Fields ML (1978) Enrichment of home-prepared
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alkali-induced transformations of
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York: The New York Academy of

41

ACKNOWLEDGEMENTS

The author expresses sincere appreciation to Dr.
Carole A. Z. Harbers, Associate Professor of Foods and
Nutrition and Major Professor, for encouragement,
patience, and advice throughout the writer's graduate
study; and for guidance in the preparation of this man-
uscript. The author wishes to thank the following who
helped during the course of this study: Dr. Daniel Y. C.
Fung, Professor of Animal Sciences and Industry, Dr.
Meredith F. Smith, Assistant Professor of Foods and
Nutrition, and Dr. Clyde E. Wassom, Professor of Agron-
omy, for technical advice and for serving on the writer's
committee; Mr. Ronald Wasserstein, Instructor of Sta-
tistics, Washburn University, for the experimental design
and analysis of data; Dr. Leniel H. Harbers, Professor of
Animal Sciences and Industry, and Miss Kathlean J.
Zeleznak, Research Assistant, Department of Grain Science
and Industry, for technical assistance; and Mr. and Mrs.
Jerry Williams, friends of the author, for assistance in
typing the manuscript.

42

APPENDIX

43

Table 6. Raw data for corn: thiamin* and pH* measurements,

source

Mexi co

mean

Kansas

mean

Venezuel a

mean

Mfl/g

thiamin

5.12
5.00
5.01
5.06
5.06

4.95
4.88
4.98
4.98
4.94

4.13
4.12
4.03
3.98
4.06

6.24
6.25
6.31
6.33
6.28

6.

29

6.

27

6.

23

6.

22

6.

25

6,

,24

6,

.24

6

.36

6

.40

6

.31

*Mean of two samples/replication
a Thiochrome assay

44

Table 7. Raw data for arepas: chemical*, microbiological*,
pH*, and moisture* measurements.

source

Mexico

mean

Kansas

mean

Venezuel a

jag/q thiamin

mean

chem a

micro D

£H

%moi sture

3.67

6.01

7.09

44.00

3.93

5.91

7.41

46.15

3.95

6.53

7.41

45.35

3.81

6.64

7.23

44.55

3.84

6.27

7.28

44.26

3.30

4.88

7.62

45.01

3.52

4.99

7.69

43.50

3.56

4.97

7.78

46.70

3.36

4.92

7.65

42.80

3.44

4.94

7.69

44.68

1.99

0.93

8.38

46.80

2.11

1.08

8.45

44.00

2.35

1.03

8.47

45.35

2.07

1.09

8.44

47.40

2.13

1.03

8.43

45.89

*Mean of two samples/replication

aThiochrome assay

b

L. f ermentum assay

THIAMIN CONTENT OF THREE SOURCES OF CORN AND AREPAS
AS DETERMINED CHEMICALLY AND MICROBIOLOGICALLY

by

LAURA LEE KELLER
B.S., Kansas State University, 1979

AN ABSTRACT OF A MASTER'S THESIS

submitted in partial fulfillment of the

requirements for the degree

MASTER OF SCIENCE

Department of Foods and Nutrition

KANSAS STATE UNIVERSITY
Manhattan, Kansas

1985

ABSTRACT

The arepa, a popular corn bread in Venezuela, is a pri-
mary thiamin source for that country. Arepas are low in
thiamin due both to low thiamin content in corn and to thi-
amin destruction during arepa preparation. Accurate detei —
mination of amount of thiamin present, including amount bio-
available, is important in assessing actual thiamin contri-
bution of arepas to the Venezuelan diet. This study was
designed to investigate thiamin values of corn and thiamin
loss during arepa preparation, and to compare chemical and
microbiological thiamin assays.

Thiamin amounts were chemically determined by the thio-
chrome method for corns from Mexico, Kansas, and Venezuela.
Homemade arepas were prepared from each source of corn, then
thiamin amounts were determined both chemically (using the
thiochrome method) and mi crobiol ogi cal ly (using Lactobacillus
fermentum ). Changes in pH values during arepa preparation
and total moisture were determined.

Data were analyzed using a split-plot design ANOVA.
When F-values were significant, Duncan's Multiple-Range Test
was used to determine differences between specific means.

Among sources, differences (P<0.001) were found for
thiamin amounts in corn and arepas (both chemical and micro-
biological measurements). Both within and among sources,
differences (P<0.05) were found for % thiamin loss during
arepa preparation, increase in pH from corn to arepa, and

differences in arepa thiamin values determined chemically
and microbiological ly. The microbiological method gave
higher thiamin values for Mexican and Kansan arepas, and
lower thiamin values for Venezuelan arepas when compared to
the chemical method.

Among sources during arepa preparation, the larger the
increase in alkalinity, the greater the loss of thiamin. For
thiamin measurements (both methods) of corn and arepas, the
Mexican corn had the highest values, and the Venezuelan corn
yielded the lowest values.

•